Analyzing bacteria without culturing

ABSTRACT

The invention generally relates to conducting an assay on a sample that isolates a pathogen from the sample and allows for analysis of the pathogen with minimal (i.e., at most 24 hrs of culturing) or no culturing of the pathogen. In certain embodiments, the invention provides methods for identifying a pathogen from a sample that involve obtaining a sample including a pathogen, conducting an assay that isolates the pathogen from the sample, culturing the isolated pathogen for at most about 24 hrs, and analyzing the pathogen.

RELATED APPLICATIONS

The present application is a continuation of U.S. non-provisional patentapplication Ser. No. 15/496,790, filed Apr. 25, 2017, which is acontinuation of U.S. non-provisional patent application Ser. No.13/091,548, filed Apr. 21, 2011, which claims the benefit of andpriority to U.S. provisional patent application Ser. No. 61/326,588,filed Apr. 21, 2010, the contents of each of which are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The invention generally relates to conducting an assay on a sample thatisolates a pathogen from the sample and allows for analysis of thepathogen with minimal (i.e., at most 24 hrs of culturing) or noculturing of the pathogen.

BACKGROUND

Blood-borne pathogens are a significant healthcare problem. A delayed orimproper diagnosis of a bacterial infection can result in sepsis—aserious, and often deadly, inflammatory response to the infection.Sepsis is the 10^(th) leading cause of death in the United States. Earlydetection of bacterial infections in blood is the key to preventing theonset of sepsis. Traditional methods of detection and identification ofblood-borne infection include blood culture and antibioticsusceptibility assays. Those methods typically require culturing cells,which can be expensive and can take as long as 72 hours. Often, septicshock will occur before cell culture results can be obtained.

Alternative methods for detection of pathogens, particularly bacteria,have been described by others. Those methods include molecular detectionmethods, antigen detection methods, and metabolite detection methods.Molecular detection methods, whether involving hybrid capture orpolymerase chain reaction (PCR), require high concentrations of purifiedDNA for detection. Both antigen detection and metabolite detectionmethods also require a relatively large amount of bacteria and have highlimit of detection (usually >10⁴ CFU/mL), thus requiring an enrichmentstep prior to detection. This incubation/enrichment period is intendedto allow for the growth of bacteria and an increase in bacterial cellnumbers to more readily aid in identification. In many cases, a seriesof two or three separate incubations is needed to isolate the targetbacteria. However, such enrichment steps require a significant amount oftime (e.g., at least a few days to a week) and can potentiallycompromise test sensitivity by killing some of the cells sought to bemeasured.

There is a need for methods for isolating target analytes, such asbacteria, from a sample, such as a blood sample, without an additionalenrichment step. There is also a need for methods of isolating targetanalytes that are fast and sensitive in order to provide data forpatient treatment decisions in a clinically relevant time frame.

SUMMARY

The present invention provides methods and devices for isolatingpathogens in a biological sample. The invention allows the rapiddetection of pathogen at very low levels in the sample; thus enablingearly and accurate detection and identification of the pathogen. Sincemethods of the invention are able to isolate pathogens at very lowlevels, methods of the invention reduce or eliminate the culturing stepthat is typically associated with pathogen analysis and allow for morerapid analysis of the isolated pathogen. In this manner, methods of theinvention provide data for patient treatment decisions in a clinicallyrelevant time frame.

In certain aspects, methods of the invention involve obtaining a sampleincluding a pathogen, conducting an assay that isolates the pathogenfrom the sample, culturing the isolated pathogen for at most about 24hrs, and analyzing the pathogen. The invention may be carried out withmagnetic particles having a target-specific binding moiety. Methods ofthe invention may involve introducing magnetic particles including atarget-specific binding moiety to a body fluid sample in order to createa mixture, incubating the mixture to allow the particles to bind to atarget, applying a magnetic field to capture target/magnetic particlecomplexes on a surface, and washing with a wash solution that reducesparticle aggregation, thereby isolating target/magnetic particlecomplexes. A particular advantage of methods of the invention is forcapture and isolation of bacteria and fungi directly from blood samplesat low concentrations that are present in many clinical samples (as lowas 1 CFU/ml of bacteria in a blood sample).

The target-specific binding moiety will depend on the target to becaptured. The moiety may be any capture moiety known in the art, such asan antibody, an aptamer, a nucleic acid, a protein, a receptor, a phageor a ligand. In particular embodiments, the target-specific bindingmoiety is an antibody. In certain embodiments, the antibody is specificfor bacteria. In other embodiments, the antibody is specific for fungi.

The target analyte refers to the target that will be captured andisolated by methods of the invention. The target may be bacteria, fungi,protein, a cell, a virus, a nucleic acid, a receptor, a ligand, or anymolecule known in the art. In certain embodiments, the target is apathogenic bacteria. In other embodiments, the target is a gram positiveor gram negative bacteria. Exemplary bacterial species that may becaptured and isolated by methods of the invention include E. coli,Listeria, Clostridium, Mycobacterium, Shigella, Borrelia, Campylobacter,Bacillus, Salmonella, Staphylococcus, Enterococcus, Pneumococcus,Streptococcus, and a combination thereof.

Methods of the invention may be performed with any type of magneticparticle. Magnetic particles generally fall into two broad categories.The first category includes particles that are permanently magnetizable,or ferromagnetic; and the second category includes particles thatdemonstrate bulk magnetic behavior only when subjected to a magneticfield. The latter are referred to as magnetically responsive particles.Materials displaying magnetically responsive behavior are sometimesdescribed as superparamagnetic. However, materials exhibiting bulkferromagnetic properties, e.g., magnetic iron oxide, may becharacterized as superparamagnetic when provided in crystals of about 30nm or less in diameter. Larger crystals of ferromagnetic materials, bycontrast, retain permanent magnet characteristics after exposure to amagnetic field and tend to aggregate thereafter due to strongparticle-particle interaction. In certain embodiments, the particles aresuperparamagnetic beads. In other embodiments, the magnetic particlesinclude at least 70% superparamagnetic beads by weight. In certainembodiments, the superparamagnetic beads are from about 100 nm to about250 nm in diameter. In certain embodiments, the magnetic particle is aniron-containing magnetic particle. In other embodiments, the magneticparticle includes iron oxide or iron platinum.

In certain embodiments, the incubating step includes incubating themixture in a buffer that inhibits cell lysis. In certain embodiments,the buffer includes Tris(hydroximethyl)-aminomethane hydrochloride at aconcentration of between about 50 mM and about 100 mM, preferably about75 mM. In other embodiments, methods of the invention further includeretaining the magnetic particles in a magnetic field during the washingstep. Methods of the invention may be used with any body fluid.Exemplary body fluids include blood, sputum, serum, plasma, urine,saliva, sweat, and cerebral spinal fluid.

Another aspect of the invention provides methods for identifying apathogen from a sample that involve obtaining a sample comprising apathogen, conducting an assay that isolates the pathogen from thesample, and analyzing the pathogen without first culturing the pathogen.

Another aspect of the invention provides methods for identifying apathogen from a sample that involve introducing magnetic particlescomprising a target-specific binding moiety to a sample in order tocreate a mixture, incubating the mixture to allow the particles to bindto a pathogen in the sample, applying a magnetic field to isolatepathogen/magnetic particle complexes from the sample, culturing theisolated pathogen for at most about 24 hrs, and analyzing the pathogen.

DETAILED DESCRIPTION

The invention generally relates to conducting an assay on a sample thatisolates a pathogen from the sample and allows for analysis of thepathogen with minimal (i.e., at most 24 hrs of culturing) or noculturing of the pathogen. In certain embodiments, methods of theinvention involve obtaining a sample including a pathogen, conducting anassay that isolates the pathogen from the sample, culturing the isolatedpathogen for at most about 24 hrs, and analyzing the pathogen. In otherembodiments, culturing is for at most about 23 hrs, for at most about 22hrs. for at most about 21 hrs, for at most about 20 hrs, for at mostabout 15 hrs, for at most about 10 hrs, for at most about 9 hrs, for atmost about 8 hrs, for at most about 7 hrs, for at most about 6 hrs, forat most about 5 hrs, for at most about 4 hrs, for at most about 3 hrs,for at most about 2 hrs, or for at most about 1 hr. In particularembodiments, culturing is for less than 1 hr, for example, about 45minutes, about 30 minutes, about 15 minutes, about 10 minutes, or forless than 10 minutes. In particular embodiments, culturing is completelyeliminated and the isolated bacteria is analyzed directly without anyculturing, i.e., culturing is eliminated.

In certain aspects, methods of the invention involve introducingmagnetic particles including a target-specific binding moiety to a bodyfluid sample in order to create a mixture, incubating the mixture toallow the particles to bind to a target, applying a magnetic field tocapture target/magnetic particle complexes on a surface, therebyisolating target/magnetic particle complexes. Methods of the inventionmay further involve washing the mixture in a wash solution that reducesparticle aggregation. Certain fundamental technologies and principlesare associated with binding magnetic materials to target entities andsubsequently separating by use of magnet fields and gradients. Suchfundamental technologies and principles are known in the art and havebeen previously described, such as those described in Janeway(Immunobiology, 6^(th) edition, Garland Science Publishing), the contentof which is incorporated by reference herein in its entirety.

Methods of the invention involve collecting a body fluid having a targetanalyte in a container, such as a blood collection tube (e.g.,VACUTAINER, test tube specifically designed for venipuncture,commercially available from Becton, Dickinson and company). In certainembodiments, a solution is added that prevents or reduces aggregation ofendogenous aggregating factors, such as heparin in the case of blood.

A body fluid refers to a liquid material derived from, for example, ahuman or other mammal. Such body fluids include, but are not limited to,mucus, blood, plasma, serum, serum derivatives, bile, phlegm, saliva,sweat, amniotic fluid, mammary fluid, urine, sputum, and cerebrospinalfluid (CSF), such as lumbar or ventricular CSF. A body fluid may also bea fine needle aspirate. A body fluid also may be media containing cellsor biological material. In particular embodiments, the fluid is blood.

Methods of the invention may be used to detect any target analyte. Thetarget analyte refers to the substance in the sample that will becaptured and isolated by methods of the invention. The target may bebacteria, fungi, a protein, a cell (such as a cancer cell, a white bloodcell a virally infected cell, or a fetal cell circulating in maternalcirculation), a virus, a nucleic acid (e.g., DNA or RNA), a receptor, aligand, a hormone, a drug, a chemical substance, or any molecule knownin the art. In certain embodiments, the target is a pathogenic bacteria.In other embodiments, the target is a gram positive or gram negativebacteria. Exemplary bacterial species that may be captured and isolatedby methods of the invention include E. coli, Listeria, Clostridium,Mycobacterium, Shigella, Borrelia, Campylobacter, Bacillus, Salmonella,Staphylococcus, Enterococcus, Pneumococcus, Streptococcus, and acombination thereof. A particular advantage of methods of the inventionis for capture and isolation of bacteria and fungi directly from bloodsamples at low concentrations that are present in many clinical samples(as low as 1 CFU/ml of bacteria in a blood sample).

The sample is then mixed with magnetic particles having a particularmagnetic moment and also including a target-specific binding moiety togenerate a mixture that is allowed to incubate such that the particlesbind to a target in the sample, such as a bacterium in a blood sample.The mixture is allowed to incubate for a sufficient time to allow forthe particles to bind to the target analyte. The process of binding themagnetic particles to the target analytes associates a magnetic momentwith the target analytes, and thus allows the target analytes to bemanipulated through forces generated by magnetic fields upon theattached magnetic moment.

In general, incubation time will depend on the desired degree of bindingbetween the target analyte and the magnetic beads (e.g., the amount ofmoment that would be desirably attached to the target), the amount ofmoment per target, the amount of time of mixing, the type of mixing, thereagents present to promote the binding and the binding chemistry systemthat is being employed. Incubation time can be anywhere from about 5seconds to a few days. Exemplary incubation times range from about 10seconds to about 2 hours. Binding occurs over a wide range oftemperatures, generally between 15° C. and 40° C.

Methods of the invention may use any magnetic particulars. In certainembodiments, methods of the invention are performed with magneticparticle having a magnetic moment that allows for isolation of as low as1 CFU/ml of bacteria in the sample. Production of magnetic particles isshown for example in Giaever (U.S. Pat. No. 3,970,518), Senyi et al.(U.S. Pat. No. 4,230,685), Dodin et al. (U.S. Pat. No. 4,677,055),Whitehead et al. (U.S. Pat. No. 4,695,393), Benjamin et al. (U.S. Pat.No. 5,695,946), Giaever (U.S. Pat. No. 4,018,886), Rembaum (U.S. Pat.No. 4,267,234), Molday (U.S. Pat. No. 4,452,773), Whitehead et al. (U.S.Pat. No. 4,554,088), Forrest (U.S. Pat. No. 4,659,678), Liberti et al.(U.S. Pat. No. 5,186,827), Own et al. (U.S. Pat. No. 4,795,698), andLiberti et al. (WO 91/02811), the content of each of which isincorporated by reference herein in its entirety.

Magnetic particles generally fall into two broad categories. The firstcategory includes particles that are permanently magnetizable, orferromagnetic; and the second category includes particles thatdemonstrate bulk magnetic behavior only when subjected to a magneticfield. The latter are referred to as magnetically responsive particles.Materials displaying magnetically responsive behavior are sometimesdescribed as superparamagnetic. However, materials exhibiting bulkferromagnetic properties, e.g., magnetic iron oxide, may becharacterized as superparamagnetic when provided in crystals of about 30nm or less in diameter. Larger crystals of ferromagnetic materials, bycontrast, retain permanent magnet characteristics after exposure to amagnetic field and tend to aggregate thereafter due to strongparticle-particle interaction. In certain embodiments, the particles aresuperparamagnetic beads. In certain embodiments, the magnetic particleis an iron containing magnetic particle. In other embodiments, themagnetic particle includes iron oxide or iron platinum.

In certain embodiments, the magnetic particles include at least about10% superparamagnetic beads by weight, at least about 20%superparamagnetic beads by weight, at least about 30% superparamagneticbeads by weight, at least about 40% superparamagnetic beads by weight,at least about 50% superparamagnetic beads by weight, at least about 60%superparamagnetic beads by weight, at least about 70% superparamagneticbeads by weight, at least about 80% superparamagnetic beads by weight,at least about 90% superparamagnetic beads by weight, at least about 95%superparamagnetic beads by weight, or at least about 99%superparamagnetic beads by weight. In a particular embodiment, themagnetic particles include at least about 70% superparamagnetic beads byweight.

In certain embodiments, the superparamagnetic beads are less than 100 nmin diameter. In other embodiments, the superparamagnetic beads are about150 nm in diameter, are about 200 nm in diameter, are about 250 nm indiameter, are about 300 nm in diameter, are about 350 nm in diameter,are about 400 nm in diameter, are about 500 nm in diameter, or are about1000 nm in diameter. In a particular embodiment, the superparamagneticbeads are from about 100 nm to about 250 nm in diameter.

In certain embodiments, the particles are beads (e.g., nanoparticles)that incorporate magnetic materials, or magnetic materials that havebeen functionalized, or other configurations as are known in the art. Incertain embodiments, nanoparticles may be used that include a polymermaterial that incorporates magnetic material(s), such as nanometalmaterial(s). When those nanometal material(s) or crystal(s), such asFe₃O₄, are superparamagnetic, they may provide advantageous properties,such as being capable of being magnetized by an external magnetic field,and demagnetized when the external magnetic field has been removed. Thismay be advantageous for facilitating sample transport into and away froman area where the sample is being processed without undue beadaggregation.

One or more or many different nanometal(s) may be employed, such asFe₃O₄, FePt, or Fe, in a core-shell configuration to provide stability,and/or various others as may be known in the art. In many applications,it may be advantageous to have a nanometal having as high a saturatedmoment per volume as possible, as this may maximize gradient relatedforces, and/or may enhance a signal associated with the presence of thebeads. It may also be advantageous to have the volumetric loading in abead be as high as possible, for the same or similar reason(s). In orderto maximize the moment provided by a magnetizable nanometal, a certainsaturation field may be provided. For example, for Fe₃O₄superparamagnetic particles, this field may be on the order of about 0.3T.

The size of the nanometal containing bead may be optimized for aparticular application, for example, maximizing moment loaded upon atarget, maximizing the number of beads on a target with an acceptabledetectability, maximizing desired force-induced motion, and/ormaximizing the difference in attached moment between the labeled targetand non-specifically bound targets or bead aggregates or individualbeads. While maximizing is referenced by example above, otheroptimizations or alterations are contemplated, such as minimizing orotherwise desirably affecting conditions.

In an exemplary embodiment, a polymer bead containing 80 wt % Fe₃O₄superparamagnetic particles, or for example, 90 wt % or highersuperparamagnetic particles, is produced by encapsulatingsuperparamagnetic particles with a polymer coating to produce a beadhaving a diameter of about 250 nm.

Magnetic particles for use with methods of the invention have atarget-specific binding moiety that allows for the particles tospecifically bind the target of interest in the sample. Thetarget-specific moiety may be any molecule known in the art and willdepend on the target to be captured and isolated. Exemplarytarget-specific binding moieties include nucleic acids, proteins,ligands, antibodies, aptamers, and receptors.

In particular embodiments, the target-specific binding moiety is anantibody, such as an antibody that binds a particular bacterium. Generalmethodologies for antibody production, including criteria to beconsidered when choosing an animal for the production of antisera, aredescribed in Harlow et al. (Antibodies, Cold Spring Harbor Laboratory,pp. 93-117, 1988). For example, an animal of suitable size such asgoats, dogs, sheep, mice, or camels are immunized by administration ofan amount of immunogen, such the target bacteria, effective to producean immune response. An exemplary protocol is as follows. The animal isinjected with 100 milligrams of antigen resuspended in adjuvant, forexample Freund's complete adjuvant, dependent on the size of the animal,followed three weeks later with a subcutaneous injection of 100micrograms to 100 milligrams of immunogen with adjuvant dependent on thesize of the animal, for example Freund's incomplete adjuvant. Additionalsubcutaneous or intraperitoneal injections every two weeks withadjuvant, for example Freund's incomplete adjuvant, are administereduntil a suitable titer of antibody in the animal's blood is achieved.Exemplary titers include a titer of at least about 1:5000 or a titer of1:100,000 or more, i.e., the dilution having a detectable activity. Theantibodies are purified, for example, by affinity purification oncolumns containing protein G resin or target-specific affinity resin.

The technique of in vitro immunization of human lymphocytes is used togenerate monoclonal antibodies. Techniques for in vitro immunization ofhuman lymphocytes are well known to those skilled in the art. See, e.g.,Inai, et al., Histochemistry, 99(5):335 362, May 1993; Mulder, et al.,Hum. Immunol., 36(3):186 192, 1993; Harada, et al., J. Oral Pathol.Med., 22(4):145 152, 1993; Stauber, et al., J. Immunol. Methods,161(2):157 168, 1993; and Venkateswaran, et al., Hybridoma, 11(6) 729739, 1992. These techniques can be used to produce antigen-reactivemonoclonal antibodies, including antigen-specific IgG, and IgMmonoclonal antibodies.

Any antibody or fragment thereof having affinity and specific for thebacteria of interest is within the scope of the invention providedherein. Immunomagnetic beads against Salmonella are provided in Vermuntet al. (J. Appl. Bact. 72:112, 1992). Immunomagnetic beads againstStaphylococcus aureus are provided in Johne et al. (J. Clin. Microbiol.27:1631, 1989). Immunomagnetic beads against Listeria are provided inSkjerve et al. (Appl. Env. Microbiol. 56:3478, 1990). Immunomagneticbeads against Escherichia coli are provided in Lund et al. (J. Clin.Microbiol. 29:2259, 1991).

Methods for attaching the target-specific binding moiety to the magneticparticle are known in the art. Coating magnetic particles withantibodies is well known in the art, see for example Harlow et al.(Antibodies, Cold Spring Harbor Laboratory, 1988), Hunter et al.(Immunoassays for Clinical Chemistry, pp. 147-162, eds., ChurchillLivingston, Edinborough, 1983), and Stanley (Essentials in Immunologyand Serology, Delmar, pp. 152-153, 2002). Such methodology can easily bemodified by one of skill in the art to bind other types oftarget-specific binding moieties to the magnetic particles. Certaintypes of magnetic particles coated with a functional moiety arecommercially available from Sigma-Aldrich (St. Louis, Mo.).

In certain embodiments, a buffer solution is added to the sample alongwith the magnetic beads. An exemplary buffer includesTris(hydroximethyl)-aminomethane hydrochloride at a concentration ofabout 75 mM. It has been found that the buffer composition, mixingparameters (speed, type of mixing, such as rotation, shaking etc., andtemperature) influence binding. It is important to maintain osmolalityof the final solution (e.g., blood+buffer) to maintain high labelefficiency. In certain embodiments, buffers used in methods of theinvention are designed to prevent lysis of blood cells, facilitateefficient binding of targets with magnetic beads and to reduce formationof bead aggregates. It has been found that the buffer solutioncontaining 300 mM NaCl, 75 mM Tris-HCl pH 8.0 and 0.1% Tween 20 meetsthese design goals.

Without being limited by any particular theory or mechanism of action,it is believed that sodium chloride is mainly responsible formaintaining osmolality of the solution and for the reduction ofnon-specific binding of magnetic bead through ionic interaction.Tris(hydroximethyl)-aminomethane hydrochloride is a well establishedbuffer compound frequently used in biology to maintain pH of a solution.It has been found that 75 mM concentration is beneficial and sufficientfor high binding efficiency. Likewise, Tween 20 is widely used as a milddetergent to decrease nonspecific attachment due to hydrophobicinteractions. Various assays use Tween 20 at concentrations ranging from0.01% to 1%. The 0.1% concentration appears to be optimal for theefficient labeling of bacteria, while maintaining blood cells intact.

An alternative approach to achieve high binding efficiency whilereducing time required for the binding step is to use static mixer, orother mixing devices that provide efficient mixing of viscous samples athigh flow rates, such as at or around 5 mL/min. In one embodiment, thesample is mixed with binding buffer in ratio of, or about, 1:1, using amixing interface connector. The diluted sample then flows through amixing interface connector where it is mixed with target-specificnanoparticles. Additional mixing interface connectors providing mixingof sample and antigen-specific nanoparticles can be attached downstreamto improve binding efficiency. The combined flow rate of the labeledsample is selected such that it is compatible with downstreamprocessing.

After binding of the magnetic particles to the target analyte in themixture to form target/magnetic particle complexes, a magnetic field isapplied to the mixture to capture the complexes on a surface. Componentsof the mixture that are not bound to magnetic particles will not beaffected by the magnetic field and will remain free in the mixture.Methods and apparatuses for separating target/magnetic particlecomplexes from other components of a mixture are known in the art. Forexample, a steel mesh may be coupled to a magnet, a linear channel orchannels may be configured with adjacent magnets, or quadrapole magnetswith annular flow may be used. Other methods and apparatuses forseparating target/magnetic particle complexes from other components of amixture are shown in Rao et al. (U.S. Pat. No. 6,551,843), Liberti etal. (U.S. Pat. No. 5,622,831), Hatch et al. (U.S. Pat. No. 6,514,415),Benjamin et al. (U.S. Pat. No. 5,695,946), Liberti et al. (U.S. Pat. No.5,186,827), Wang et al. (U.S. Pat. No. 5,541,072), Liberti et al. (U.S.Pat. No. 5,466,574), and Terstappen et al. (U.S. Pat. No. 6,623,983),the content of each of which is incorporated by reference herein in itsentirety.

In certain embodiments, the magnetic capture is achieved at highefficiency by utilizing a flow-through capture cell with a number ofstrong rare earth bar magnets placed perpendicular to the flow of thesample. When using a flow chamber with flow path cross-section 0.5 mm×20mm (h×w) and 7 bar NdFeB magnets, the flow rate could be as high as 5mL/min or more, while achieving capture efficiency close to 100%.

The above described type of magnetic separation produces efficientcapture of a target analyte and the removal of a majority of theremaining components of a sample mixture. However, such a process mayproduce a sample that contains a percent of magnetic particles that arenot bound to target analytes, as well as non-specific target entities.Non-specific target entities may for example be bound at a much lowerefficiency, for example 1% of the surface area, while a target ofinterest might be loaded at 50% or nearly 100% of the available surfacearea or available antigenic cites. However, even 1% loading may besufficient to impart force necessary for trapping in a magnetic gradientflow cell or sample chamber.

The presence of magnetic particles that are not bound to target analytesand non-specific target entities on the surface that includes thetarget/magnetic particle complexes may interfere with the ability tosuccessfully detect the target of interest. The magnetic capture of theresulting mix, and close contact of magnetic particles with each otherand bound targets, result in the formation of aggregate that is hard todispense and which might be resistant or inadequate for subsequentprocessing or analysis steps. In order to remove magnetic particles thatare not bound to target analytes and non-specific target entities,methods of the invention may further involve washing the surface with awash solution that reduces particle aggregation, thereby isolatingtarget/magnetic particle complexes from the magnetic particles that arenot bound to target analytes and non-specific target entities. The washsolution minimizes the formation of the aggregates.

Methods of the invention may use any wash solution that imparts a netnegative charge to the magnetic particle that is not sufficient todisrupt interaction between the target-specific moiety of the magneticparticle and the target analyte. Without being limited by any particulartheory or mechanism of action, it is believed that attachment of thenegatively charged molecules in the wash solution to magnetic particlesprovides net negative charge to the particles and facilitates dispersalof non-specifically aggregated particles. At the same time, the netnegative charge is not sufficient to disrupt strong interaction betweenthe target-specific moiety of the magnetic particle and the targetanalyte (e.g., an antibody-antigen interaction). Exemplary solutionsinclude heparin, Tris-HCl, Tris-borate-EDTA (TBE), Tris-acetate-EDTA(TAE), Tris-cacodylate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid), PBS (phosphatebuffered saline), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid), MES(2-N-morpholino)ethanesulfonic acid), Tricine(N-(Tri(hydroximethyl)methyl)glycine), and similar buffering agents. Incertain embodiments, only a single wash cycle is performed. In otherembodiments, more than one wash cycle is performed.

In particular embodiments, the wash solution includes heparin. Forembodiments in which the body fluid sample is blood, the heparin alsoreduces probability of clotting of blood components after magneticcapture. The bound targets are washed with heparin-containing buffer 1-3times to remove blood components and to reduce formation of aggregates.

Since methods of the invention are able to isolate pathogens at very lowlevels, methods of the invention reduce or eliminate the culturing stepthat is typically associated with pathogen analysis and allow for morerapid analysis of the isolated pathogen. Reduced culturing refers toculturing for at most about 24 hrs, for example, for at most about 23hrs, for at most about 22 hrs. for at most about 21 hrs, for at mostabout 20 hrs, for at most about 15 hrs, for at most about 10 hrs, for atmost about 9 hrs, for at most about 8 hrs, for at most about 7 hrs, forat most about 6 hrs, for at most about 5 hrs, for at most about 4 hrs,for at most about 3 hrs, for at most about 2 hrs, or for at most about 1hr. In particular embodiments, culturing is for less than 1 hr, forexample, about 45 minutes, about 30 minutes, about 15 minutes, about 10minutes, or for less than 10 minutes. In particular embodiments,culturing is completely eliminated and the isolated bacteria is analyzeddirectly without any culturing, i.e., culturing is eliminated.

The target may be analyzed by a multitude of existing technologies, suchas miniature NMR, Polymerase Chain Reaction (PCR), mass spectrometry,fluorescent labeling and visualization using microscopic observation,fluorescent in situ hybridization (FISH), growth-based antibioticsensitivity tests, and variety of other methods that may be conductedwith purified target without significant contamination from other samplecomponents. In one embodiment, isolated bacteria are lysed with achaotropic solution, and DNA is bound to DNA extraction resin. Afterwashing of the resin, the bacterial DNA is eluted and used inquantitative RT-PCR to detect the presence of a specific species,and/or, subclasses of bacteria.

In another embodiment, captured bacteria is removed from the magneticparticles to which they are bound and the processed sample is mixed withfluorescent labeled antibodies specific to the bacteria or fluorescentGram stain. After incubation, the reaction mixture is filtered through0.2 μm to 1.0 μm filter to capture labeled bacteria while allowingmajority of free beads and fluorescent labels to pass through thefilter. Bacteria is visualized on the filter using microscopictechniques, e.g. direct microscopic observation, laser scanning or otherautomated methods of image capture. The presence of bacteria is detectedthrough image analysis. After the positive detection by visualtechniques, the bacteria can be further characterized using PCR orgenomic methods.

Detection of bacteria of interest can be performed by use of nucleicacid probes following procedures which are known in the art. Suitableprocedures for detection of bacteria using nucleic acid probes aredescribed, for example, in Stackebrandt et al. (U.S. Pat. No.5,089,386), King et al. (WO 90/08841), Foster et al. (WO 92/15883), andCossart et al. (WO 89/06699), each of which is hereby incorporated byreference.

A suitable nucleic acid probe assay generally includes sample treatmentand lysis, hybridization with selected probe(s), hybrid capture, anddetection. Lysis of the bacteria is necessary to release the nucleicacid for the probes. The nucleic acid target molecules are released bytreatment with any of a number of lysis agents, including alkali (suchas NaOH), guanidine salts (such as guanidine thiocyanate), enzymes (suchas lysozyme, mutanolysin and proteinase K), and detergents. Lysis of thebacteria, therefore, releases both DNA and RNA, particularly ribosomalRNA and chromosomal DNA both of which can be utilized as the targetmolecules with appropriate selection of a suitable probe. Use of rRNA asthe target molecule(s), may be advantageous because rRNAs constitute asignificant component of cellular mass, thereby providing an abundanceof target molecules. The use of rRNA probes also enhances specificityfor the bacteria of interest, that is, positive detection withoutundesirable cross-reactivity which can lead to false positives or falsedetection.

Hybridization includes addition of the specific nucleic acid probes. Ingeneral, hybridization is the procedure by which two partially orcompletely complementary nucleic acids are combined, under definedreaction conditions, in an anti-parallel fashion to form specific andstable hydrogen bonds. The selection or stringency of thehybridization/reaction conditions is defined by the length and basecomposition of the probe/target duplex, as well as by the level andgeometry of mis-pairing between the two nucleic acid strands. Stringencyis also governed by such reaction parameters as temperature, types andconcentrations of denaturing agents present and the type andconcentration of ionic species present in the hybridization solution.

The hybridization phase of the nucleic acid probe assay is performedwith a single selected probe or with a combination of two, three or moreprobes. Probes are selected having sequences which are homologous tounique nucleic acid sequences of the target organism. In general, afirst capture probe is utilized to capture formed hybrid molecules. Thehybrid molecule is then detected by use of antibody reaction or by useof a second detector probe which may be labelled with a radioisotope(such as phosphorus-32) or a fluorescent label (such as fluorescein) orchemiluminescent label.

Detection of bacteria of interest can also be performed by use of PCRtechniques. A suitable PCR technique is described, for example, inVerhoef et al. (WO 92/08805). Such protocols may be applied directly tothe bacteria captured on the magnetic beads. The bacteria is combinedwith a lysis buffer and collected nucleic acid target molecules are thenutilized as the template for the PCR reaction.

For detection of the selected bacteria by use of antibodies, isolatedbacteria are contacted with antibodies specific to the bacteria ofinterest. As noted above, either polyclonal or monoclonal antibodies canbe utilized, but in either case have affinity for the particularbacteria to be detected. These antibodies, will adhere/bind to materialfrom the specific target bacteria. With respect to labeling of theantibodies, these are labeled either directly or indirectly with labelsused in other known immunoassays. Direct labels may include fluorescent,chemiluminescent, bioluminescent, radioactive, metallic, biotin orenzymatic molecules. Methods of combining these labels to antibodies orother macromolecules are well known to those in the art. Examplesinclude the methods of Hijmans, W. et al. (1969), Clin. Exp. Immunol. 4,457-, for fluorescein isothiocyanate, the method of Goding, J. W.(1976), J. Immunol. Meth. 13, 215-, for tetramethylrhodamineisothiocyanate, and the method of Ingrall, E. (1980), Meth. in Enzymol.70, 419-439 for enzymes.

These detector antibodies may also be labeled indirectly. In this casethe actual detection molecule is attached to a secondary antibody orother molecule with binding affinity for the anti-bacteria cell surfaceantibody. If a secondary antibody is used it is preferably a generalantibody to a class of antibody (IgG and IgM) from the animal speciesused to raise the anti-bacteria cell surface antibodies. For example,the second antibody may be conjugated to an enzyme, either alkalinephosphatase or to peroxidase. To detect the label, after the bacteria ofinterest is contacted with the second antibody and washed, the isolatedcomponent of the sample is immersed in a solution containing achromogenic substrate for either alkaline phosphatase or peroxidase. Achromogenic substrate is a compound that can be cleaved by an enzyme toresult in the production of some type of detectable signal which onlyappears when the substrate is cleaved from the base molecule. Thechromogenic substrate is colorless, until it reacts with the enzyme, atwhich time an intensely colored product is made. Thus, material from thebacteria colonies adhered to the membrane sheet will become an intenseblue/purple/black color, or brown/red while material from other colonieswill remain colorless. Examples of detection molecules includefluorescent substances, such as 4-methylumbelliferyl phosphate, andchromogenic substances, such as 4-nitrophenylphosphate,3,3′,5,5′-tetramethylbenzidine and2,2′-azino-di-[3-ethelbenz-thiazoliane sulfonate (6)]. In addition toalkaline phosphatase and peroxidase, other useful enzymes includeβ-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase,α-mannosidase, galactose oxidase, glucose oxidase and hexokinase.

Detection of bacteria of interest using NMR may be accomplished asfollows. In the use of NMR as a detection methodology, in which a sampleis delivered to a detector coil centered in a magnet, the target ofinterest, such as a magnetically labeled bacterium, may be delivered bya fluid medium, such as a fluid substantially composed of water. In sucha case, the magnetically labeled target may go from a region of very lowmagnetic field to a region of high magnetic field, for example, a fieldproduced by an about 1 to about 2 Tesla magnet. In this manner, thesample may traverse a magnetic gradient, on the way into the magnet andon the way out of the magnet. As may be seen via equations 1 and 2below, the target may experience a force pulling into the magnet in thedirection of sample flow on the way into the magnet, and a force intothe magnet in the opposite direction of flow on the way out of themagnet. The target may experience a retaining force trapping the targetin the magnet if flow is not sufficient to overcome the gradient force.m dot (del B)=F  Equation 1v _(t) =−F/(6*p*n*r)  Equation 2where n is the viscosity, r is the bead diameter, F is the vector force,B is the vector field, and m is the vector moment of the bead.

Magnetic fields on a path into a magnet may be non-uniform in thetransverse direction with respect to the flow into the magnet. As such,there may be a transverse force that pulls targets to the side of acontainer or a conduit that provides the sample flow into the magnet.Generally, the time it takes a target to reach the wall of a conduit isassociated with the terminal velocity and is lower with increasingviscosity. The terminal velocity is associated with the drag force,which may be indicative of creep flow in certain cases. In general, itmay be advantageous to have a high viscosity to provide a higher dragforce such that a target will tend to be carried with the fluid flowthrough the magnet without being trapped in the magnet or against theconduit walls.

Newtonian fluids have a flow characteristic in a conduit, such as around pipe, for example, that is parabolic, such that the flow velocityis zero at the wall, and maximal at the center, and having a paraboliccharacteristic with radius. The velocity decreases in a direction towardthe walls, and it is easier to magnetically trap targets near the walls,either with transverse gradients force on the target toward the conduitwall, or in longitudinal gradients sufficient to prevent target flow inthe pipe at any position. In order to provide favorable fluid drag forceto keep the samples from being trapped in the conduit, it may beadvantageous to have a plug flow condition, wherein the fluid velocityis substantially uniform as a function of radial position in theconduit.

When NMR detection is employed in connection with a flowing sample, thedetection may be based on a perturbation of the NMR water signal causedby a magnetically labeled target (Sillerud et al., JMR (Journal ofMagnetic Resonance), vol. 181, 2006). In such a case, the sample may beexcited at time 0, and after some delay, such as about 50 ms or about100 ms, an acceptable measurement (based on a detected NMR signal) maybe produced. Alternatively, such a measurement may be producedimmediately after excitation, with the detection continuing for someduration, such as about 50 ms or about 100 ms. It may be advantageous todetect the NMR signal for substantially longer time durations after theexcitation.

By way of example, the detection of the NMR signal may continue for aperiod of about 2 seconds in order to record spectral information athigh-resolution. In the case of parabolic or Newtonian flow, theperturbation excited at time 0 is typically smeared because the wateraround the perturbation source travels at different velocity, dependingon radial position in the conduit. In addition, spectral information maybe lost due to the smearing or mixing effects of the differential motionof the sample fluid during signal detection. When carrying out an NMRdetection application involving a flowing fluid sample, it may beadvantageous to provide plug-like sample flow to facilitate desirableNMR contrast and/or desirable NMR signal detection.

Differential motion within a flowing Newtonian fluid may havedeleterious effects in certain situations, such as a situation in whichspatially localized NMR detection is desired, as in magnetic resonanceimaging. In one example, a magnetic object, such as a magneticallylabeled bacterium, is flowed through the NMR detector and its presenceand location are detected using MRI techniques. The detection may bepossible due to the magnetic field of the magnetic object, since thisfield perturbs the magnetic field of the fluid in the vicinity of themagnetic object. The detection of the magnetic object is improved if thefluid near the object remains near the object. Under these conditions,the magnetic perturbation may be allowed to act longer on any givenvolume element of the fluid, and the volume elements of the fluid soaffected will remain in close spatial proximity. Such a stronger, morelocalized magnetic perturbation will be more readily detected using NMRor MRI techniques.

If a Newtonian fluid is used to carry the magnetic objects through thedetector, the velocity of the fluid volume elements will depend onradial position in the fluid conduit. In such a case, the fluid near amagnetic object will not remain near the magnetic object as the objectflows through the detector. The effect of the magnetic perturbation ofthe object on the surrounding fluid may be smeared out in space, and thestrength of the perturbation on any one fluid volume element may bereduced because that element does not stay within range of theperturbation. The weaker, less-well-localized perturbation in the samplefluid may be undetectable using NMR or MRI techniques.

Certain liquids, or mixtures of liquids, exhibit non-parabolic flowprofiles in circular conduits. Such fluids may exhibit non-Newtonianflow profiles in other conduit shapes. The use of such a fluid may proveadvantageous as the detection fluid in an application employing anNMR-based detection device. Any such advantageous effect may beattributable to high viscosity of the fluid, a plug-like flow profileassociated with the fluid, and/or other characteristic(s) attributed tothe fluid that facilitate detection. As an example, a shear-thinningfluid of high viscosity may exhibit a flow velocity profile that issubstantially uniform across the central regions of the conduitcross-section. The velocity profile of such a fluid may transition to azero or very low value near or at the walls of the conduit, and thistransition region may be confined to a very thin layer near the wall.

Not all fluids, or all fluid mixtures, are compatible with the NMRdetection methodology. In one example, a mixture of glycerol and watercan provide high viscosity, but the NMR measurement is degraded becauseseparate NMR signals are detected from the water and glycerol moleculesmaking up the mixture. This can undermine the sensitivity of the NMRdetector. In another example, the non-water component of the fluidmixture can be chosen to have no NMR signal, which may be achieved byusing a perdeuterated fluid component, for example, or using aperfluorinated fluid component. This approach may suffer from the lossof signal intensity since a portion of the fluid in the detection coildoes not produce a signal.

Another approach may be to use a secondary fluid component thatconstitutes only a small fraction of the total fluid mixture. Such alow-concentration secondary fluid component can produce an NMR signalthat is of negligible intensity when compared to the signal from themain component of the fluid, which may be water. It may be advantageousto use a low-concentration secondary fluid component that does notproduce an NMR signal in the detector. For example, a perfluorinated orperdeuterated secondary fluid component may be used. The fluid mixtureused in the NMR detector may include one, two, or more than twosecondary components in addition to the main fluid component. The fluidcomponents employed may act in concert to produce the desired fluid flowcharacteristics, such as high-viscosity and/or plug flow. The fluidcomponents may be useful for providing fluid characteristics that areadvantageous for the performance of the NMR detector, for example byproviding NMR relaxation times that allow faster operation or highersignal intensities.

A non-Newtonian fluid may provide additional advantages for thedetection of objects by NMR or MRI techniques. As one example, theobjects being detected may all have substantially the same velocity asthey go through the detection coil. This characteristic velocity mayallow simpler or more robust algorithms for the analysis of thedetection data. As another example, the objects being detected may havefixed, known, and uniform velocity. This may prove advantageous indevices where the position of the detected object at later times isneeded, such as in a device that has a sequestration chamber orsecondary detection chamber down-stream from the NMR or MRI detectioncoil, for example.

In an exemplary embodiment, sample delivery into and out of a 1.7 Tcylindrical magnet using a fluid delivery medium containing 0.1% to 0.5%Xanthan gum in water was successfully achieved. Such delivery issuitable to provide substantially plug-like flow, high viscosity, suchas from about 10 cP to about 3000 cP, and good NMR contrast in relationto water. Xanthan gum acts as a non-Newtonian fluid, havingcharacteristics of a non-Newtonian fluid that are well know in the art,and does not compromise NMR signal characteristics desirable for gooddetection in a desirable mode of operation.

In certain embodiments, methods of the invention are useful for directdetection of bacteria from blood. Such a process is described here.Sample is collected in sodium heparin tube by venipuncture, acceptablesample volume is about 1 mL to 10 mL. Sample is diluted with bindingbuffer and superparamagnetic particles having target-specific bindingmoieties are added to the sample, followed by incubation on a shakingincubator at 37° C. for about 30 min to 120 min. Alternative mixingmethods can also be used. In a particular embodiment, sample is pumpedthrough a static mixer, such that reaction buffer and magnetic beads areadded to the sample as the sample is pumped through the mixer. Thisprocess allows for efficient integration of all components into a singlefluidic part, avoids moving parts and separate incubation vessels andreduces incubation time.

Capture of the labeled targets allows for the removal of bloodcomponents and reduction of sample volume from 30 mL to 5 mL. Thecapture is performed in a variety of magnet/flow configurations. Incertain embodiments, methods include capture in a sample tube on ashaking platform or capture in a flow-through device at flow rate of 5mL/min, resulting in total capture time of 6 min.

After capture, the sample is washed with wash buffer including heparinto remove blood components and free beads. The composition of the washbuffer is optimized to reduce aggregation of free beads, whilemaintaining the integrity of the bead/target complexes.

The detection method is based on a miniature NMR detector tuned to themagnetic resonance of water. When the sample is magnetically homogenous(no bound targets), the NMR signal from water is clearly detectable andstrong. The presence of magnetic material in the detector coil disturbsthe magnetic field, resulting in reduction in water signal. One of theprimary benefits of this detection method is that there is no magneticbackground in biological samples which significantly reduces therequirements for stringency of sample processing. In addition, since thedetected signal is generated by water, there is a built-in signalamplification which allows for the detection of a single labeledbacterium.

This method provides for isolation and detection of as low as or evenlower than 1 CFU/ml of bacteria in a blood sample.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Sample

Blood samples from healthy volunteers were spiked with clinicallyrelevant concentrations of bacteria (1-10 CFU/mL) including bothlaboratory strains and clinical isolates of the bacterial species mostfrequently found in bloodstream infections.

Example 2

Antibody Preparation

In order to generate polyclonal, pan-Gram-positive bacteria-specificIgG, a goat was immunized by first administering bacterial antigenssuspended in complete Freund's adjuvant intra lymph node, followed bysubcutaneous injection of bacterial antigens in incomplete Freund'sadjuvant in 2 week intervals. The antigens were prepared for antibodyproduction by growing bacteria to exponential phase (OD₆₀₀=0.4−0.8).Following harvest of the bacteria by centrifugation, the bacteria wasinactivated using formalin fixation in 4% formaldehyde for 4 hr at 37°C. After 3 washes of bacteria with PBS (15 min wash, centrifugation for20 min at 4000 rpm) the antigen concentration was measured using BCAassay and the antigen was used at 1 mg/mL for immunization. In order togenerate Gram-positive bacteria-specific IgG, several bacterial specieswere used for inoculation: Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecium and Enterococcus fecalis.

The immune serum was purified using affinity chromatography on a proteinG sepharose column (GE Healthcare), and reactivity was determined usingELISA. Antibodies cross-reacting with Gram-negative bacteria and fungiwere removed by absorption of purified IgG with formalin-fixedGram-negative bacteria and fungi. The formalin-fixed organisms wereprepared similar to as described above and mixed with IgG. Afterincubation for 2 hrs at room temperature, the preparation wascentrifuged to remove bacteria. Final antibody preparation was clarifiedby centrifugation and used for the preparation of antigen-specificmagnetic beads.

Example 3 Preparation of Antigen-Specific Magnetic Beads

Superparamagnetic beads were synthesized by encapsulating iron oxidenanoparticles (5-15 nm diameter) in a latex core and labeling with goatIgG. Ferrofluid containing nanoparticles in organic solvent wasprecipitated with ethanol, nanoparticles were resuspended in aqueoussolution of styrene and surfactant Hitenol BC-10, and emulsified usingsonication. The mixture was allowed to equilibrate overnight withstirring and filtered through 1.2 and 0.45 μm filters to achieve uniformmicelle size. Styrene, acrylic acid and divynilbenzene were added incarbonate buffer at pH 9.6. The polymerization was initiated in amixture at 70° C. with the addition of K₂S₂O₈ and the reaction wasallowed to complete overnight. The synthesized particles were washed 3times with 0.1% SDS using magnetic capture, filtered through 1.2, 0.8,and 0.45 μm filters and used for antibody conjugation.

The production of beads resulted in a distribution of sizes that may becharacterized by an average size and a standard deviation. In the caseof labeling and extracting of bacteria from blood, the average size foroptimal performance was found to be between 100 and 350 nm, for examplebetween 200 nm to 250 nm.

The purified IgG were conjugated to prepared beads using standardchemistry. After conjugation, the beads were resuspended in 0.1% BSAwhich is used to block non-specific binding sites on the bead and toincrease the stability of bead preparation.

Example 4 Labeling of Rare Cells Using Excess of Magnetic Nanoparticles

Bacteria, present in blood during blood-stream infection, weremagnetically labeled using the superparamagnetic beads prepared inExample 3 above. The spiked samples as described in Example 1 werediluted 3-fold with a Tris-based binding buffer and target-specificbeads, followed by incubation on a shaking platform at 37° C. for up to2 hr. After incubation, the labeled targets were magnetically separatedfollowed by a wash step designed to remove blood products. See example 5below.

Example 5 Magnetic Capture of Bound Bacteria

Blood including the magnetically labeled target bacteria and excess freebeads were injected into a flow-through capture cell with a number ofstrong rare earth bar magnets placed perpendicular to the flow of thesample. With using a flow chamber with flow path cross-section 0.5 mm×20mm (h×w) and 7 bar NdFeB magnets, a flow rate as high as 5 mL/min wasachieved. After flowing the mixture through the channel in the presenceof the magnet, a wash solution including heparin was flowed through thechannel. The bound targets were washed with heparin-containing bufferone time to remove blood components and to reduce formation of magneticparticle aggregates. In order to effectively wash bound targets, themagnet was removed and captured magnetic material was resuspended inwash buffer, followed by re-application of the magnetic field andcapture of the magnetic material in the same flow-through capture cell.

Removal of the captured labeled targets was possible after movingmagnets away from the capture chamber and eluting with flow of buffersolution.

What is claimed is:
 1. A method for analyzing a pathogen in a sample,the method comprising: providing a vessel that contains an unwashed bodyfluid sample; introducing to the vessel: magnetic particles comprising apathogen-binding moiety, and a buffer that promotes binding of theparticles to pathogen; binding the magnetic particles to a pathogen inthe sample; flowing the pathogen-bound magnetic particles and unwashedsample from the vessel into a fluidic device and through a channel inthe fluidic device comprising a magnet; applying a magnetic field toisolate the pathogen from the sample; and analyzing the pathogen,wherein the pathogen is selected from the group consisting of abacterium, a fungus, and a virus.
 2. The method according to claim 1,further comprising washing the magnetic particles with a wash solutionthat reduces particle aggregation.
 3. The method according to claim 1,wherein the particles are superparamagnetic particles.
 4. The methodaccording to claim 3, wherein the particles have a diameter from about100 nm to about 250 nm.
 5. The method according to claim 4, wherein theparticles comprise at least about 65% magnetic material by weight. 6.The method according to claim 1, wherein the pathogen-binding moiety isan antibody.
 7. The method according to claim 6, wherein the antibodycomprises a detectable label.
 8. The method according to claim 1,wherein the body fluid is blood.
 9. The method according to claim 8,wherein the pathogen is a blood-borne pathogen.
 10. The method accordingto claim 1, wherein the identifying step comprises one selected from thegroup consisting of sequencing nucleic acid derived from the pathogenand amplifying nucleic acid derived from the pathogen.
 11. The method ofclaim 1, wherein the buffer comprises sodium chloride.
 12. The method ofclaim 11, wherein said buffer further comprisestris(hydroxymethyl)aminomethane hydrochloride and polysorbate
 20. 13.The method of claim 12, wherein the buffer comprisestris(hydroxymethyl)aminomethane hydrochloride at a concentration ofabout 75 mM.
 14. The method of claim 1, wherein the introducing stepproduces a mixture of sample:buffer at a ratio of about 1:1.
 15. Themethod of claim 1, wherein the fluidic device is a microfluidic device.16. The method of claim 1, wherein the flowing step comprises flowingthe sample toward a magnet.
 17. A method for detecting a pathogen in asample, the method comprising: providing a vessel that contains a bodyfluid sample; introducing to the sample in the vessel: magneticparticles comprising a pathogen-binding moiety, and a buffer thatpromotes binding of the particles to the pathogen; binding the magneticparticles to a pathogen in the sample; flowing the pathogen-boundmagnetic particles and unwashed sample from the vessel into a fluidicdevice and through a channel in the fluidic device comprising a magnet;applying a magnetic field to isolate the pathogen from the sample; anddetecting the pathogen, wherein the pathogen is selected from the groupconsisting of a bacterium, a fungus, and a virus.
 18. The methodaccording to claim 17, wherein the body fluid is blood.
 19. The methodaccording to claim 18, wherein the pathogen is a blood-borne pathogen.